The present invention concerns the use of agonists and antagonists of the peripheral-type benzodiazepine receptors (PTBRs). More particularly, the invention concerns the use of PTBR agonists and antagonists (including PTBR ligands) in the diagnosis and treatment of cardiac hypertrophy and other circulatory conditions.
In response to hormonal, physiological, hemodynamic and pathological stimuli, adult ventricular muscle cells can adapt to increased workloads through the activation of a hypertrophic process. This process is characterized by an increase in the contractile protein content of cardiac muscle cells without a proliferative response because the adult cardiomyocyte is terminally differentiated and has lost its ability to divide. Cardiac growth during the hypertrophic process therefore results primarily from an increase in protein content per individual cardiomyocyte, with little or no change in cell number. The acquisition of the cardiac hypertrophic phenotype is in part dependent upon the activation of cardiac muscle gene program.
In addition to the induction of specific contractile protein components, ventricular hypertrophy is also characterized by alterations in the expression of certain non-contractile proteins, such as atrial natriuretic peptide (ANP, also known as ANF). During embryonic development, the ANP gene is expressed in both the atrium and the ventricle. However, shortly after birth ANP expression is down regulated in the ventricle and expression is mainly confined to the atrium. Following induction of hypertrophy, ANP is reexpressed in the ventriculum. Thus, ANP expression can be considered to be a non-contractile protein marker of cardiac ventricular hypertrophy.
Ventricular hypertrophy is initially a compensatory mechanism by which the heart is attempting to counteract the effects of conditions like pressure overload, loss of contractile tissue, obstruction of blood flow, or increased peripheral demand for blood flow, all of which can be generated by a variety of physiological or pathological stimuli. In some circumstances, such as, injury or functional compromise of the heart, a typically short term, compensated hypertrophic response is desirable. Similarly, cardiac, e.g. left ventricular, hypertrophy (physiological hypertrophy) is often observed in some highly trained athletes, without any apparent cardiovascular complications. However, under some circumstances the hypertrophic response may eventually contribute to cardiac dysfunction. These circumstances include, but are not limited to, excessive hypertrophy, prolonged hypertrophy, or hypertrophy occurring in the context of toxic factors or toxic concentrations of factors that, when combined with the hypertrophic response of cardiac myocytes, result in mechanical dysfunction, electrical conduction dysfunction, loss of cardiac wall elasticity, or stimulation of fibrosis. In these cases hypertrophy is termed decompensated hypertrophy, and antagonism of cardiac hypertrophy is considered desirable. Once the transition from compensated to decompensated hypertrophy is achieved, the progression to a terminal heart failure phenotype often rapidly follows.
Heart failure affects approximately five million Americans. New cases of heart failure number about 400,000 each year. The pathophysiology of congestive heart failure is rather complex. In general, congestive heart failure is a syndrome characterized by left ventricular dysfunction, reduced exercise tolerance, impaired quality of life, and markedly shortened life expectancy. Decreased contractility of the left ventricle leads to reduced cardiac output with consequent systemic arterial and venous vasoconstriction. This vasoconstriction, which promotes the vicious cycle of further reductions of stroke volume followed by an increased elevation of vascular resistance, appears to be mediated, in part, by the renin-angiotensin system. Numerous etiologies contribute to the development of CHF, including primary diseases of, or insults to, the myocardium itself, cardiac defects, hypertension, inflammation, kidney disease and vascular disease. These conditions lead to the hypertrophy and remodeling of the cardiac ventricles which, if unchecked, ultimately reduce the mechanical performance of the heart. Forces associated with the inability of the heart to pump blood ultimately lead to the release of neurohormones like catecholamines, renin-angiotensin, aldosterone, endothelin and related factors into the circulation. It has been demonstrated that elevations in plasma levels of many of these circulating neurohoiiones may have a deleterious impact on the outcome of patients with CHF. Local production of these neurohormonal factors in the heart is believed to contribute centrally to the disease. Thus, an important therapeutic strategy has been to block this neurohormonal axis contributing to the pathogenesis of this disease.
Factors known to contribute centrally to the pathophysiology of heart disease are biosynthesized in the heart itself. These factors are produced in cardiac myocytes, fibroblasts, smooth muscle and endothelial cells, and inflammatory cells associated with the myocardium. For example, the heart has been shown to contain its own renin-angiotensin system. Blockade of the cardiac renin-angiotensin system is believed to contribute significantly to the therapeutic efficacy of the therapeutic class of agents known as angiotensin converting enzyme (ACE) inhibitors.
The heart also produces other factors including, but not limited to, endothelins, bradykinin, adrenomedullin, tumor necrosis factor, transforming growth factors, and natriuretic peptides. While there are successful therapeutic approaches based on the modulation of these secondary factors, there is a need for devising different strategies that directly modulate the cardiac hypertrophic response.
Thus, there is a great interest in trying to understand the mechanisms that induce and control ventricular hypertrophy and indeed to dissect the transition from compensated to decompensated hypertrophy. There are several physiological stimuli that will induce a hypertrophic response in isolated cardiomyocytes such as endothelin-1, TGF-xcex2 and angiotensin II. Additionally, the xcex1 adrenergic agonist phenylephrine is a well-characterized and potent inducer of hypertrophy in isolated cardiomyocytes.
In the course of our functional genomic studies, the gene of a peripheral-type benzodiazepine receptor (PTBR) was found to be differentially expressed in the hearts of several rat models of heart failure. Peripheral-type benzodiazepine receptors represent a subset of the benzodiazcpine receptor family distinguished by their location outside the central nervous system (CNS). A review of PTBR""s, including the molecular structure, biological properties and possible physiological roles has been published by Zisterer and Williams, Gen. Pharmac. 29:305-314 (1997), the entire disclosure of which is hereby expressly incorporated by reference.
Ligands of PTBR""s have been known for many years and anti-depressant CNS effects of PTBR agonists (e.g. Valium) are widely known. Vagal tone has been found to decrease following intravenous administration of diazepam (Adinoff et al., Psychiatry Research 41:89-97 [1992]). There is evidence for control of cardiac vagal tone by benzodiazcpine receptors (DiMicco, Neurophaimacology 26:553-559 [1987]). PTBR ligands Ro5-4864 and PK11195, but not diazepam, have been described to depress cardiac function in an isolated working rat heart model (Edoute et al., Pharmacology 46:224-230 [1993]). Ro5-4864 has also been reported to increase coronary flow in an isolated perfused Langendorf rat heart without affecting heart rate and left ventricular contractility. PK11195 did not antagonize this vasodilatory effect (Grupp et al., Eur. J. Pharm. 143:143-147 [1987]). In an isolated rat heart preparation, diazepam induced a transient negative inotropic effect followed by a positive inotropic response. The positive inotropy was antagonized by PK11195. (Leeuwin et al., Eur. J. Pharm. 299:149-152 [1996]). Diazepam increased contractile force in Langendorf rat heart (Leeuwin et al., Arch. Int. Pharmacodyn. 326:5-12 [1993]). Ro5-4864 has been shown to have a small (20%) depressant effect on the contraction amplitude (negative inotropic effect) of human atrial strips that was not antagonized by K11195 (Shany et al., Eur.J. Pharm. 253:231-236 [1994]). In a guinea pig heart preparation Ro5-4864 decreased the duration of intracellular action potential and contractility. Diazepam was less effective and clonazepam ineffective. The effects of Ro5-4864 were reversed by PK11195 but not by a specific antagonist of the CNS BZR. (Mestre et al., Life Sciences 35:953-962 [1984]). The presence of PTBR binding sites in the hearts of dogs and humans was demonstrated in vivo by positron emission tomography using [11C]-PK11195. (Charmonneau ie al., Circulation 73:476-483 [1986]). It has also been reported that Ro5-4862 and dipyridamole can compete [3H]diazepam binding to heart tissue. Diazepam potentiates the actions of adenosine on isolated cardiac and smooth muscle and the coronary vasodilator action of adenosine in dogs. There is evidence that diazepam may be acting in a similar manner to dipyridamole by inhibiting adenosine uptake (Davies and Huston, Eur. J. Pharm. 73:209-211 [1981]).
While there are reports of various effects of diazepam and its derivatives upon heart function, these effects have been attributed to their anti-depressant effects in decreasing vagal tone and not by direct effects upon cardiomyocyte function.
There is a need for the identification of endogenous and exogenous factors that will promote or inhibit the ventricular hypertrophic phenotype. Specifically, there is a need to identify factors that are therapeutics or instrumental in the identification of therapeutics effective in the treatment of heart failure or as preventative agents for the treatment of patients at high risk of developing heart failure.
The present invention concerns the use of agonist and antagonists of the peripheral-type benzodiazepine receptors (PTBR""s), such as PTBR ligands, to induce or inhibit cardiac hypertrophy. In particular, the invention concerns the use of antagonists of the PTBR""s in the prevention or treatment of decompensated cardiac hypertrophy and eventually, heart failure. The invention also concerns the use of agonists of the PTBR""s in the management of conditions calling for increased blood flow or cardiac output, including, without limitation, injury or functional compromise of the heart, increased demand for physical exercise by athletes or by those who need extra help to improve cardiac performance as a result of a disability, existing atrio-ventricular (A-V) shunts, an acquired or inherited predisposition to cardiac contractile protein dysfunction, etc.
In one aspect, the invention concerns a method of inducing a hypertrophic response in cardiac myocytes by contacting the myocytes with an effective amount of an agonist of a peripheral-type benzodiazepine receptor (PTBR). The treatment may be performed in vitro or in vivo, and the PTBR preferably is a native receptor of a mammalian species, e.g. human, while the agonist preferably is a PTBR ligand.
In another aspect, the invention concerns a method of reducing a hypertrophic response of cardiac myocytes by contacting the myocytes with an effective amount of an antagonist of a peripheral-type benzodiazepine receptor (PTBR). Again, the treatment may be performed in vitro or in vivo, and the PTBR preferably is a native receptor of a mammalian species, e.g. human, while the antagonist preferably is a PTBR ligand.
In yet another aspect, the invention concerns a method for the treatment (including prevention) of cardiac hypertrophy by administering to a patient an effective amount of a PTBR antagonist. The cardiac hypertrophy to be treated preferably is decompensated hypertrophy, and the preferred treatment is early intervention used to prevent, reverse, or slow down the progression of this condition.
In a further aspect, the invention concerns a method for inducing compensated cardiac hypertrophy by administering to a patient in need an effective amount of a PTBR agonist. This approach is typically used in a situation where increased blood flow or pressure would be beneficial without fear of adverse consequences, such as congestive heart failure or decompensation. Hence, PTBR agonists are particularly useful in the treatment (including prevention) of conditions where a, typically short term, compensatory mechanism is desirable to respond to factors like pressure overload, loss of contractile tissue or function, obstruction of blood flow, or increased peripheral demand for blood flow or cardiac output.
In a still further aspect, the invention concerns a method of screening for a PTBR antagonist by contacting a cardiac myocyte of hypertrophic phenotype with a candidate molecule, and monitoring the reduction in hypertrophy.
In another aspect, the invention concerns a method for screening for a PTBR agonist by contacting a normal cardiac myocyte with a candidate molecule, and monitoring the appearance of hypertrophic phenotype.
In all screening assays, the candidate preferably is a molecule capable of binding to a PTBR or, in the case of PTBR antagonist candidates, to a native PTBR ligand.
In a different aspect, the invention concerns a method for the prevention of decompensated cardiac hypertrophy by administering to a patient an effective amount of a PTBR antagonist. In a preferred embodiment, the method concerns the prevention of the progression of compensated cardiac hypertrophy into decompensated cardiac hypertrophy.
In another aspect, the invention concerns a method for the treatment (including prevention) of heart failure comprising administering to a patient an effective amount of a PTBR antagonist. The heart failure may be congestive heart failure due to ischemia, drug or toxin exposure, infection, altered metabolism, genetic predisposition to altered contractile function, or other cause.
The invention further concerns a composition for the treatment (including prevention) of a cardiac disease comprising an effective amount of a PTBR antagonist or agonist, in admixture with a pharmaceutically acceptable excipient. If the composition comprises a PTBR antagonist, the cardiac disease preferably is cardiac hypertrophy regardless of the underlying mechanism. If the composition comprises a PTBR agonist, the goal preferably is to assist the patient to whom the composition is administered, in coping with a condition that calls for increased cardiac or peripheral blood flow, by inducing, under controlled conditions, compensated cardiac hypertrophy.
In yet another aspect, the invention concerns a method for diagnosing a heart disease comprising detecting an alteration in the expression level of a PTBR or an endogenous ligand thereof. The heart disease preferably is compensated or decompensated cardiac hypertrophy. Proper and timely diagnosis will enable the attending physician to customize therapeutic modalties to a patient""s cardiac disease.
In a further aspect, the invention concerns a method of treating a patient in need by administering an agonist of a PTBR followed by the administration of an antagonist of a PTBR. This method is particularly useful when initially the patient is in need of increased cardiac or peripheral blood flow, for example as a result of loss of contractile tissue or obstruction of blood flow, therefore, the administration of a PTBR agonist is desirable, but later develops or is in danger of developing decompensated cardiac hypertrophy.
In all aspects, the PTBR agonist may, for example, be a native sequence PTBR ligand or a fragment or functional subunit thereof, an organic small molecule or peptide, a polypeptide variant of a native sequence ligand, an antibody, a glycopeptide, a glycolipid, a polysaccharide, an oligosaccharide, a nucleic acid, a peptidomimetic, a pharmacological agent or a metabolite thereof, a transcriptional or translational control sequence, and the like. Similarly, the PTBR antagonist may be a polypeptide, an organic small molecule or peptide, a polypeptide variant of a native sequence ligand, an antibody, a glycopeptide, a glycolipid, a polysaccharide, an oligosaccharide, a nucleic acid, a peptidomimetic, a pharmacological agent or a metabolite thereof, a transcriptional or translational control sequence, and the like. For example, PTBR antagonists include polypeptide variants of a native sequence PTBR ligand, variants of a native sequence PTBR that retain the ability to bind an endogenous ligand but are deficient in their ability to mediate biological activity, anti-PTBR or anti-PTBR ligand antibodies, and selective inhibitors of the in vivo production of an endogenous PTBR ligand. The organic small molecules arc preferably selected from the chemical classes of benzodiazepines, isoquinoline carboxamides, imidazopyridines, 2-aryl-3-indoleacetamides, and pyrolobenzoxazepines. A particularly preferred agonist is Ro5-4864, while a particularly preferred antagonist is PK11195.
The PTBR agonist or antagonists may be administered orally, by intravenous or subcutaneous administration, or by direct infusion into the coronary vasculature, pericardial space, or cardiac tissue, on an acute or chronic or recurring basis.